Stimulation of chemotaxis by chemotactic peptides

ABSTRACT

A method of stimulating chemotaxis toward a prosthetic device is disclosed, which method comprises incorporating a chemotactic peptide of the formula 
     
         B.sup.1 -X-(AGVPGLGVG).sub.n -(AGVPGFGVG).sub.m -Y-B.sup.2 
    
     wherein 
     A is a peptide-forming residue of L-alanine; 
     P is a peptide-forming residue of L-proline; 
     G is a peptide-forming residue of glycine; 
     V is a peptide-forming residue of L-valine; 
     F is a peptide-forming residue of L-phenylalanine; 
     L is a peptide-forming residue of L-leucine; 
     B 1  is H or a biocompatible N-terminal group; 
     B 2  is OH, OB 3  where B 3  is a non-toxic metal ion, or a biocompatible C-terminal group; 
     X is GVPGFGVG, GVPGLGVG, VPGFGVG, VPGLGVG, PGFGVG, PGLGVG, GFGVG, GLGVG, FGVG, LGVG, GVG, VG, G or a covalent bond; 
     Y is AGVPGFGV, AGVPGLGV, AGVPGFG, AGVPGLG, AGVPGF, AGVPGL, AGVPG, AGVP, AGV, AG, A or a covalent bond; 
     n is an integer from 0 to 50; 
     m is an integer from 0 to 50; with the proviso that when both n and m are O, X and Y are selected so that the chemotactic peptide has at least 3 amino acid residues in the X and Y positions combined; into a surface of a prosthetic device. Prosthetic devices which have the property of enhancing invasion of fibroblasts and endothelial cells as a result of the chemotactic peptide are also disclosed.

This application is a continuation-in-part of U.S. Ser. No. 07/013,343,filed Feb. 11, 1987 now abandoned, which is a continuation of U.S.Serial No. 06/793,225, filed Oct. 31, 1985, now U.S. Pat. No. 4,693,718.

BACKGROUND OF THE INVENTION

This work was supported in part by grants from the National Institutesof Health and the Government has certain rights in the invention as aresult of this support.

1. Field of the Invention

This invention relates to stimulation of chemotaxis, particularly inrelation to prosthetic devices

2. Description of the Prior Art

Replacement of a blood vessel by a prosthetic device is an important andcommon practice in modern vascular surgery. Although some use is made ofveins or arteries taken from other portions of a patient's body, most ofsuch prosthetic devices are prepared from artificial materials that canbe prepared in a variety of sizes and stored in a sterile state readyfor use.

There are several essential properties of cardiovascular prostheticmaterials, among which are the following:

1. Retardation of thrombosis and thromboembolism (antithrombogenic);

2. Minimal harm to blood cells and minimal blood cell adhesion;

3. Long life as prosthetic inserts: and

4. High compliance with the physical and chemical properties of naturalblood vessel such as similar elastic modulus and tensile strength.

Another useful property would be a chemotactic response that inducedrapid endothelialization and invasion of connective tissue cells forvascular wall reconstruction in a manner such that the prosthesis wouldbe slowly replaced by and/or integrated into newly synthesized internalelastic lamina. None of the materials presently being used can fulfillall of these requirements.

The most commonly used fabric for blood vessel prosthesis is made fromDacron (Trademark, DuPont), a synthetic polyester fiber made frompolyethylene terephthalate. Dacron has been used in several weaves andin combination with other materials. An example of a frequently usedmaterial is the DeBakey Elastic Dacron fabric manufactured by USCI, adivision of C.R. Bard, Inc. (Cat. No. 007830). Other commonly usedmaterials are felted polyurethane and polytetrafluorethylene (Berkowitzet al, Surgery, 72, 221 (1972); Wagner et al, J. Surg. Res., 1, 53(1956); Goldfarb et al, Trans. Am. Soc. Art. Int. Org., XXIII, 268(1977)). No chemotactic substance is normally used with these materials.

Another recent development in prosthetic devices is artificial skin ofthe type disclosed in Yannas and Burke, J. Biomed. Mat. Res., 14, 65-81(1980). The artificial skin is a collagen/glycosaminoglycan (GAG)composite and had been successfully tested as full-thickness skin woundreplacements. Such membranes have effectively protected wounds frominfection and fluid loss for long periods of time without rejection andwithout requiring change or other invasive manipulation. Appropriatelydesigned artificial skin of this type has retarded wound contraction,and the artificial skin has been replaced, at least in part, by newlysynthesized connective tissue. Additional disclosure of this artificialskin is found in Yannas et al, ibid, 107-131 (1980), and Dagalakis etal, ibid, 511-528 (1980). No synthetic chemotactic substance is normallyused with these materials.

One chemotactic material that might be useful in enhancing invasion offibroblasts into such prosthetic devices is platelet-derived growthfactor (PDGF), a potent fibroblast chemo-attractant. Unfortunately, PDGFcannot be synthesized and must be obtained from platelets, making theutilization of such a material on a wide scale impractical.

Recently, a chemotactic peptide has been identified in tropoelastin andis described in U.S. Pat. No. 4,605,413. This material is a chemotacticpeptide having a 6-amino-acid repeating unit of formula APGVGV and itsactive permutation VGVAPG, in which A represents alanine, P representsproline, G represents glycine, and V represents valine. Although thismaterial readily produces chemotaxis and is a natural component of thehuman body, therefore making it particularly suitable for use in vivo,room remains for additional improvements in the field of chemotacticstimulation, for example in cell specificity and sensitivity.

Accordingly, there remains a need for an artificial and easilysynthesized chemotactic material capable of attracting fibroblasts andendothelial cells into prosthetic devices and thereby enhancing theincorporation of such devices into the regenerating natural tissue.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to provide a syntheticmaterial having chemotactic properties towards cells such as fibroblastsand endothelial cells.

It is a further object of this invention to provide a prosthetic devicewhich is readily incorporated into regenerating tissue, such as skin orblood vessel walls.

It is still another object of this invention to provide a chemotacticmaterial having stimulating activity to a greater extent than waspreviously available.

These and other objects of the invention as will hereinafter become morereadily apparent have been accomplished by providing a method ofstimulating chemotaxis, which comprises: incorporating a chemotacticpeptide of the formula

    B.sup.1 -X-(AGVPGLGVG).sub.n -(AGVPGFGVG).sub.m -Y-B.sup.2

wherein

A is a peptide-forming residue of L-alanine;

P is a peptide-forming residue of L-proline;

G is a peptide-forming residue of glycine;

V is a peptide-forming residue of L-valine;

F is a peptide-forming residue of L-phenylalanine;

L is a peptide-forming residue of L-leucine;

B¹ is H or a biocompatible N-terminal group;

B² is OH, OB³ where B³ is a non-toxic metal ion, or a biocompatibleC-terminal group:

X is GVPGFGVG, GVPGLGVG, VPGFGVG, VPGLGVG, PGFGVG, PGLGVG, GFGVG, GLGVG,FGVG, LGVG, GVG, VG, G or a covalent bond;

Y is AGVPGFGV, AGVPGLGV, AGVPGFG, AGVPGLG, AGVPGF, AGVPGL, AGVPG, AGVP,AGV, AG, A or a covalent bond;

n is an integer from 0 to 50;

m is an integer from 0 to 50; with the proviso that when both n and mare 0, X and Y are selected so that the chemotactic peptide has at least3 amino acid residues in the X and Y positions combined;

into a layer of a prosthetic device in an amount sufficient to increasechemotaxis towards said layer.

This invention also comprises chemotactic matrices and prostheticdevices prepared according to the method set forth above.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings wherein:

FIG. 1 is a graph of the chemotactic dose response of fibroblasts toAGVPGFGVG;

FIG. 2 is a graph of the chemotactic dose response of fibroblasts toGFGVGAGVP;

FIG. 3 is a graph of the chemotactic dose response of fibroblasts tohuman platelet-derived growth factor (comparison);

FIG. 4 is a graph of the chemotactic dose response of fibroblasts toVGVAPG (comparison);

FIG. 5 is a carbon-13 nuclear magnetic resonance spetra of the Phe andLeu containing nonapeptides at 25 MHz, 29° C. in DMSO-d₆ a.H-GFGVGAGVP-OH b. H-GLGVGAGVP-OH;

FIG. 6 is a phase contrast image of a typical monolayer of bovine aorticendothelial cells, which exhibit a "cobblestone" distribution pattern.After incubation with Dil-Ac-LDL, cells showed a punctate fluorescencedistribution pattern, indicative of endosomal localization of thelabeled LDL derivative internalized by cells via receptor-mediatedendocytosis. All cells were positive in fluorescent labeling withrespect to controls, with variability in cell to cell staining densitytypically present.

FIG. 7 shows bovine aortic endothelial cell migration response to theelastin repeat nonapeptides; GFGVGAGVP ₁₃.₁₃ and GLGVGAGVP . Netfibronectin migration for ₁₃. -- curve was 30 cells per h.p.f. and 46cells per h.p.f. for curve. Background migration was 38 and 95 cellsh.p.f. for the two nonapeptides respectively.

FIG. 8 shows bovine aortic endothelial cell migration in response to theelastin repeat peptide VGVAPG. Net fibronectin migration was 28 cellsper h.p.f. and background migration was 38 cells per h.p.f.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention arose as the result of investigations into thestructure of elastic fibers present in blood vessel walls and otherelastic materials, such as ligaments, present in humans and animals. Thecentral portion of the elastic fibers of vascular wall, skin, lung andligament is derived from a single protein called tropoelastin.Polypeptide sequences of tropoelastin from vascular wall have been shownby Sandberg and colleagues to contain a repeat hexapeptide(Ala-Pro-Gly-Val-Gly-Val)n, a repeat pentapeptide(Val-Pro-Gly-Val-Gly)n, and a repeat tetrapeptide (Val-Pro-Gly-Gly)n,where Ala, Pro, Val and Gly respectively represent alanine, proline,valine and glycine amino acid residues. (Peptide representations in thisapplication conform to the standard practice of writing the NH₂-terminal amino acid residue at the left of the formula and the CO₂Hterminal amino acid residue at the right.) A high polymer of thehexapeptide has been synthesized, whereby it forms cellophane-likesheets. The hexapeptide has therefore been thought to fill a structuralrole in the natural material.

However, recent investigations have indicated that this hexapeptide andpermutations of this sequence are chemotactic for fibroblasts whichsynthesize elastic fiber precursor protein in biological systems. As aresult of this discovery and related investigations into the variouspermutations of the natural material, U.S. Pat. No. 4,605,413 disclosesand claims a synthetic material based on the hexapeptide sequence.

Further investigations into tropoelastin have revealed the presence of anonapeptide that repeats four times in a single continuous sequence.Investigations into the property of materials based on this repeatingunit had indicated that synthetic materials produced having thissequence are potent chemotactic agents having an activity even higherthan synthetic materials based on the previously discovered hexapeptidesequence. At their maximum activity, the nonapeptides are as active asplatelet-derived growth factor (PDGF) at 30 ng/ml. The nonapeptidestherefore are fully as potent as the previously discovered hexapeptidesand achieve this activity at a comparable or slightly lowerconcentration. It is expected that enhanced invasion ofelastic-fiber-synthesizing fibroblasts and/or endothelial cells willoccur when a prosthetic device, designed for incorporation intoregenerating tissue, is treated by incorporating a chemotactic peptideof the formula

    B.sup.1 -X-(AGVPGLGVG).sub.n -(AGVPGFGVG).sub.m -Y-B.sup.2

wherein

A is a peptide-forming residue of L-alanine;

P is a peptide-forming residue of L-proline;

G is a peptide-forming residue of glycine;

V is a peptide-forming residue of L-valine;

F is a peptide-forming residue of L-phenylalanine;

L is a peptide-forming residue of L-leucine;

B¹ is H or a biocompatible N-terminal group;

B² is OH, OB³ where B³ is a non-toxic metal ion, or a biocompatibleC-terminal group:

X is GVPGFGVG, GVPGLGVG, VPGFGVG, VPGLGVG, PGFGVG, PGLGVG, GFGVG, GLGVG,FGVG, LGVG, GVG, VG, G or a covalent bond;

Y is AGVPGFGV, AGVPGLGV, AGVPGFG, AGVPGLG, AGVPGF, AGVPGL, AGVPG, AGVP,AGV, AG, A or a covalent bond;

n is an integer from 0 to 50;

m is an integer from 0 to 50; with the proviso that when both n and mare 0, X and Y are selected so that the chemotactic peptide has at least3 amino acid residues in the X and Y positions combined;

into a surface of the prosthetic layer. In this way the layer of theprosthetic device becomes the source of a concentration gradient of thechemotactic peptide.

Both the isolated nonamers, such as H-AGVPGFGVG-OH, and polynonapeptideshave the chemotactic property. The nonapeptide H-GFGVGAGVP-OH has beenshown to have essentially the same chemotactic activity asH-AGVPGFGVG-OH. Chemotactic activity is also expected for the otherpermutations; i.e., H-GVPGFGVGA-OH, H-VPGFGVGAG-OH, H-PGFGVGAGV-OH,H-FGVGAGVPG-OH, H-GVGAGVPGF-OH, H-VGAGVPGFG-OH, and H-GAGVPGFGV-OH. Whena polynonapeptide is present, the compound (perhaps in the form offragments derived therefrom by in vivo enzymatic action) is chemotacticregardless of the value of n or m. However, for ease of handling, valuesof n and m combined of no more than 100 are preferred since highermolecular weight compounds have limited solubility and are difficult tohandle. Preferred values of n and m are each from 1 to 10, with valuesof about 5 being most preferred.

Preferably, B¹ and B² are H and OH, respectively. It can be seen fromthe above formula, that the peptides of the present invention can bemade up of a repeating nonapeptide associated with n in the aboveformula or a repeating peptide associated with m in the above formula.When n is 0 and m is 1 or greater, the resulting compounds are the sameas those in U.S. Ser. No. 793,225, now U.S. Pat. No. 4,693,718. When nand m are each 0, X and Y are selected so that the resulting chemotacticpeptide has at least 3 amino acid residues in the X and Y positions.Still more preferably, when n and m are 0, X and Y are selected so thatthe resulting peptide has at least 5 amino acids in the X and Ypositions combined. More preferably, the selected chemotactic peptidehas from 5 to 40 amino acid residues. When the peptide has 5 aminoacids, it is preferably GFGVG. Most preferably, the chemotactic peptidehas 9 amino acid residues. It is also preferred that n is at least 1. Itis also preferred that if m is greater than 0, n must be at least 1 or Xor Y must be a residue containing a leucine residue. In a most preferredembodiment, n is 1 and m is 0.

It will be noted that polynonapeptides can be synthesized using any ofthe nonapeptide "monomers" listed above. Thus, polynonapeptidesgenerally will have the structure B¹ -(repeating unit)_(n) -B² where B¹and B² represent end groups which are discussed later. The repeatingunit can be any of the permutations of the nonamers listed above. Infact, if the chemotactic peptide is not synthesized from nonapeptide"monomers" but rather is synthesized by sequential addition of aminoacids to a growing peptide (such as in an automatic peptide synthesizeror by use of an artificial gene) the designation of a repeating unit issomewhat arbitrary. For example, the peptideH-GFGVGAGVPGFGVGAGVPGFGVGAGVPGF-OH can be considered to consist of anyof the following repeating units and end groups, among others:H-(GFGVGAGVP)₃ -GF-OH, H-G-(FGVGAGVPG)₃ -F-OH, H-GF-(GVGAGVPGF)₃ -OH,H-GFG(VGAGVPGFG)₂ -VGAGVPGF-OH, H-GFGV-(GAGVPGFGV)₂ -GAGVPGF-OH, orH-GFGVG-(AGVPGFGVG)₂ -AGVPGF-OH.

Synthesis of the chemotactic peptide is straightforward and easilyaccomplished by a peptide chemist. The resulting peptides generally havethe structure B¹ -(repeating unit)_(n) (repeating unit)_(m) -B² where B¹and B² represent any chemically compatible end group on the amino andcarboxyl ends of the molecule, respectively, and n and m are from 0 toabout 50. When B¹ is H, B² is OH, n=1 and m=0, the compound is anonapeptide. When n or m is greater than 1, the compound is apolynonapeptide (often referred to herein as a polypeptide). It ispossible that one or more amino acid residue or segment of amino acidresidues not present in the normal polynonapeptide sequence may beinterspersed within a polynonapeptide chain so long as the chemotacticcharacter of the resulting molecule is not completely disrupted. Asclearly indicated by the formula and by the following discussion, theinvention encompasses incorporation of a nonamer or polynonapeptide intoa larger peptide chain in which B¹ and B² represent the remainder of thelarger peptide chain.

Other examples of terminal B¹ and B2 end groups include portions of therepeating peptide units themselves with free amino or carboxylic acidgroups or salts thereof, free amino or carboxylic acid groups or salts(especially alkali metal salts), and peptide or amino acid units thathave retained a blocking group that was present during synthesis of thepolypeptide or that have a biocompatible group added after formation ofthe polypeptide. Examples of blocking groups include t-butyloxycarbonyl,formyl, and acetyl for the amino end of the molecule and esters, such asmethyl esters, as well as amides, such as the amides of ammonia andmethyl amine, for the acid end of the molecule. The end groups are notcritical and can be any organic or inorganic group that does not destroythe chemotactic properties of the polypeptide or conferbio-incompatibility to the molecule as a whole. The term biologicallycompatible as used in this application means that the component inquestion will not harm the organism in which it is implanted to such adegree that implantation is as harmful as or more harmful than theneeded prosthetic device.

Methods of preparing polypeptide polymers have been disclosed in Rapakaand Urry, Int. J. Peptide Protein Res., 11, 97 (1978), Urry et al,Biochemistry, 13, 609 (1974), and Urry et al, J. Mol. Biol., 96, 101(1975), which are herein incorporated by reference. The synthesis ofthese peptides is straightforward and can be easily modified to any ofthe peptides disclosed herein. The following summary, which is not to beconsidered limiting, is an example of the general method of synthesizingthe polypeptides.

The first step in the formation of a polynonapeptide of the inventionusually is synthesis of a nonapeptide monomer. Any of the classicalmethods of producing peptide molecules may be used in synthesizing thebuilding blocks of the polymers of the present invention. For examplesynthesis can be carried out by classical solution techniques startingfrom the Cterminal amino acid as a benzyl (Bzl) ester ptosylate. Eachsuccessive amino acid is then coupled to the growing peptide chain bymeans of its water-soluble carbodiimide and 1-hydroxybenzotriazole. Atypically used carbodiimide is1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI).During the coupling reaction the amino group is protected. Theprotecting group is then removed after condensation has taken place. Asuitable protecting group is tert-butyloxycarbonyl (Boc), which caneasily be removed by trifluoroacetic acid.

The first product obtained in the synthesis of the hexapeptide monomeris a protected nonapeptide, such asBoc-L.Ala-Gly-L.Val-L.Pro-Gly-L.Phe-Gly-L.Val-Gly-OBzl. This protectedmonomer is converted into the reactive monomer by, for example,replacement of the benzyl ester with the p-nitrophenyl ester, forexample by effectively exchanging with p-nitrophenyl trifluoroacetate,and removal of the Boc protecting group. The resulting reactive monomeris polymerized, in the presence of a base such as triethylamine asnecessary, to give the polypeptide. A blocking group, such as H-Val-OMemay be added at the conclusion of the polymerization reaction to convertthe remaining reactive p-nitrophenyl esters to non-reactive terminalgroups if desired.

Since all of the amino acids present in the polypeptides of theinvention have corresponding DNA codons, the polypeptides can also beproduced by genetic engineering using synthetic genes containing codonsthat correspond t the desired amino acid sequence.

When a modified chemical structure is desired, as, for example, whenchemical cross-linking between two chains of polynonapeptide or betweena polynonapeptide chain and a peptide-forming part of the structure of aprosthetic device will be carried out, side-group-blocked lysine orglutamic acid (or another amino acid with a protected side group capableof forming a cross-link after the protecting group is removed) may beutilized in place of one of the normal amino acids that is present inthe polypeptide chain. A synthesis of a chemically cross-linkedpolypentapeptide of similar structure is disclosed in U.S. Pat. No.4,187,852, which is herein incorporated by reference.

It is not necessary for the chemotactic peptide of the invention to becovalently attached to the surface toward which chemotaxis is beingstimulated. It is sufficient that the peptide be present at the surfaceor layer. Therefore, the phrase "incorporating into a surface or layer"as used in this application encompasses all methods of applying achemotactic peptide of this invention to a surface, whether thatapplication results in chemical bonding or not. For example, solutionsor suspensions containing the peptide can be painted on the surface of aprosthetic device or a device can be submerged in a solution of thechemotactic peptide and be made to swell taking in the chemotacticpeptide and then can be made to contract expelling the water but leavingthe chemotactic peptide within the matrix of the device.

It is also possible to form covalent bonds between the chemotacticpeptide and the prosthetic device. For example, during the synthesis ofa chemotactic peptide as described above, various intermediates areproduced which have reactive carboxy or amino terminals. Many of theprosthetic devices which are intended for incorporation intoregenerating tissue are prepared from collagen or related materials andtherefore contain free amino acid functional groups, such as amino orcarboxylic acid groups. Peptide bonds can easily be formed between suchfunctional groups in the prosthetic device and reactive intermediatessuch as those described above.

The type of prosthetic device which can be used in conjunction with thepresent invention is not limited, since the chemotactic property isrelated to the peptide and not to the prosthetic device itself. It ispreferred, however, that the prosthetic device be one which is intendedfor incorporation into regenerating tissue, such as an artificial veinor artery or artificial skin. Publications which disclose variousprosthetic devices useful for forming artificial skin or blood vesselwalls are listed in the section of this application entitled BackgroundOf the Invention, and these publications are herein incorporated byreference. Two particularly preferred embodiments of the presentinvention involve using the chemotactic polypeptide with acollagen/glycosaminoglycan composite material as an artificial skin, asdescribed in U.S. Pat. No. 4,280,954, and with biocompatible artificialmaterials based on polypeptides as described in U.S. Pat. No. 4,187,852:U.S. Pat. application Ser. No. 308,091, filed Oct. 2, 1981; and U.S.Pat. application No. 452,801, filed Dec. 23, 1982, all of which areherein incorporated by reference. These are peptide-containingmaterials, and the chemotactic polypeptide may readily be attached bycovalent bonding into such materials by the methods described above.However, as also previously indicated, covalent bonding is not necessaryand indeed is not preferred since the chemotactic property is alsoexhibited when the chemotactic peptide is merely present on the surfaceor in the pores of a prosthetic material. Prosthetic devices havingsurfaces comprising other structural peptides are also preferred overprosthetic devices having other types of surfaces, although other typesof surfaces, such as Dacron, and other synthetic fibers, arespecifically included. Examples include natural materials such tendonsor ligaments (for example, those transferred from one location toanother within the same body) and synthetic or semi-synthetic materials.Semi-synthetic materials are those derived by manipulation of naturalmaterials, such as collagen.

The amount of chemotactic peptide which is required for a particularprosthetic device can easily be determined by simple experimentation.Generally, quite low concentrations of the chemotactic peptide arerequired. For example, doping of a non-chemotactic surface to producelow concentrations of 0.1 nM to 100 nM of a chemotactic peptide at thesurface will be sufficient. Generally, from 10⁻⁹ to 10⁻³ millimoles ofnonamer or repeating unit of a polynonapeptide per 100 cm² of surface issufficient for this purpose. It is preferred to produce a concentrationof the chemotactic nonamer of from 10⁻¹⁰ to 10⁻⁶ M, preferably 10⁻⁹ to10⁻⁷ M at the responsive cell.

Alternatively or additionally, a 2-component synthetic bioelastomercomprising the chemotactic peptide of this invention and the elasticpolypentapeptide or polytetrapeptide of U.S. Pat. No. 4,187,852 wouldact as a chemotactic elastic biopolymer which could be utilized for avariety of purposes. It is also possible to use the chemotactic peptideof this invention in a system involving natural crosslinking ofsynthetic bioelastomers, as is described in U.S. Pat. application Ser.No. 533,524, now U.S. Pat. No. 4,589,882, which is herein incorporatedby reference. That application discloses bioelastomers which areenzymatically cross-linked by lysyl oxidase.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples, which are provided herein for purposes ofillustration only and are not intended to be limiting unless otherwisespecified.

EXAMPLE 1

The chemotactic response of fibroblasts to two nonapeptides of theinvention was measured using the techniques described below.

Materials and Methods Source of Chemicals and Materials

Dulbecco's Modified Eagle Medium (DMEM), nonessential amino acids,L-glutamine, fetal bovine serum, penicillin-streptomycin solution:GIBCO, Chagrin, Ohio. Trypsin 1-300: ICN, Cleveland, Ohio. Ethylenediaminetetraacetic acid (EDTA)--sodium salt and gelatin: Sigma, St.Louis, Mo. Human serum albumin (HSA): American National Red Cross,Washington, D.C. Hematoxylin: Harleco, Gibbstown, NJ. Platelet-derivedgrowth factor (PDGF): Calbiochem., San Diego, CA. Polycarbonatemembranes: Nucleopore Corp., Pleasanton, CA. Cellulose nitratemembranes: Millipore Corporation, Bedford, MA.

Cells

Passage-two fetal bovine ligamentum nuchae fibroblast cell cultures weregrown to confluency in 75 cm² plastic tissue culture flasks withDulbecco's Modified Eagle (DME) medium containing 10% fetal calf serum,2 mM L-glutamine, 0.1 mM nonessential amino acids, penicillin (100U/ml), streptomycin (100 μg/ml). These passage-two cell cultures wereobtained from passage-one cell cultures provided by R. M. Senior and R.B. Mecham (Washington University, St. Louis, MO). Ligamentum nuchaeexplants were obtained from fetal calves greater than 180 days ingestational age. Because of the possibility of platelet derived factorsbeing present in the fetal calf serum, the cells were harvested after a48 hour fast. At one to two days past confluency, the cells weredispersed by trypsin (0.025% trypsin, 0.1% EDTA in phosphate bufferedsaline, pH 7.4) and washed two times in a second DME medium containing 2mM glutamine, 0.1 mM nonessential amino acids and 1 mg/ml human serumalbumin. Cell concentration was determined with a hemocytometer andadjusted to a final concentration of 1.5×10⁵ cells/ml. The cells wereused as is for the chemotaxis assay.

Chemotaxis Assay

The chemotaxis experiment used a 30 hole multi-blindwell plate as anadaptation of the modified Boyden chamber. The upper and lowercompartments were separated by an 8 μm polycarbonate membranesuperimposed on a 0.45 μm cellulose nitrate membrane. The 8 μm membranewas pretreated with 5% gelatin to enhance fibroblast attachment. Thelower compartment contained 0.24 ml of the second DME medium plus/minuschemotactic peptides while the upper one had 0.37 ml of the second DMEmedium with fibroblasts. In the checkerboard assay, peptide was alsoadded to the upper compartment. The filled plate was placed in ahumidified incubator at 37° C. with 5% CO₂ -air for 6 hours, after whichthe cell suspension was aspirated and the membranes were recovered,fixed in ethanol, stained in Harris' Alum hematoxylin, dehydrated in agraded series of propanol and cleared in xylene. Quantitation of celllocomotion to the area between the two membranes was done with a lightmicroscope, fitted with a bright field objective and eye piece grid, at400 ×. There were 3 membrane pairs per experimental condition and 5randomly chosen fields were examined per pair. Every experiment had anegative control, i.e. medium alone, and a positive control, i.e.platelet-derived growth factor, PDGF, in the lower compartment.

Number of cells per high power field are expressed in FIGS. 1 through 3and in Tables I and II as the net number of cells that migrated throughthe polycarbonate membrane. The number of cells moving with medium alonein the lower compartment was subtracted from the number of cells movingin response to one of the peptides.

                  TABLE I                                                         ______________________________________                                        Checkboard Analysis of AGVPGFGVG with Fibroblasts                                    Peptide concentration above filters (M)                                       0     10.sup.-10 10.sup.-9 10.sup.-8                                   ______________________________________                                        Pep- 0       (10)0 ±                                                                            -2 ± 1.1                                                                            -2 ± 0.5                                                                           -3 ± 0.9                             tide         1.3                                                              con. 10.sup.-10                                                                            6 ±  -3 ± 1.0                                                                            -4 ± 0.8                                                                            2 ± 1.8                             be-          1.3                                                              low  10.sup.-9                                                                             25 ±  3 ± 1.7                                                                            -4 ± 0.6                                                                           -2 ± 1.1                             filt-        1.6                                                              ers  10.sup.-8                                                                             9 ±   5 ± 1.6                                                                             5 ± 1.2                                                                           -2 ± 1.1                             (M)          2.3                                                              ______________________________________                                         Results are expressed as means ± S.E.M. where n = 15. Positive control     PDGF at 60 μg/ml = 21.                                                

                  TABLE II                                                        ______________________________________                                        Checkboard Analysis of GFGVGAGVP with Fibroblasts                                    Peptide concentration above filters (M)                                       0     10.sup.-10 10.sup.-9 10.sup.-8                                   ______________________________________                                        Pep- 0       (9)0 ±                                                                             1 ± 1.0                                                                              3 ± 1.2                                                                           -2 ± 1.4                             tide         1.0                                                              con. 10.sup.-10                                                                            13 ± -1 ± 0.8                                                                            -3 ± 0.7                                                                           -1 ± 0.8                             be-          1.6                                                              low  10.sup.-9                                                                             45 ± 7 ± 1.8                                                                             -2 ± 1.0                                                                           -1 ± 1.1                             filt-        3.4                                                              ers  10.sup.-8                                                                             21 ± 12 ± 1.6                                                                             7 ± 1.5                                                                             0 ± 1.50                           (M)          1.5                                                              ______________________________________                                         Results are expressed as mean ± S.E.M. where n = 15. Positive control,     PDGF at 30 μg/ml = 59.                                                

Peptide Synthesis

Elemental analyses were carried out by MicAnal, Tuscon Az. All aminoacids are of L-configuration except for glycine.Tertiary-butyloxcarbonyl (Boc) amino acids and amino acid benzyl esters(Bzl) were purchased from Bachem, Inc., Torrance, CA. Thin-layerchromatography was performed on silica gel plates obtained from Whatman,Inc., NJ and mentioned as R_(f) values in different solvent systems.R_(f) ¹ chloroform (C), methanol (M), acetic acid (A), 95:5:3; R_(f) ²CMA (85:15:3); R_(f) ³ CMA (75:25:3); R_(f) ⁴ ethyl acetate, aceticacid, ethanol (90:10:10). The hexapeptide was synthesized and purifiedas outlined in Senior et al., J. Cell Biol. 99: 870-874 (1984). Thesynthesis of the nonapeptides is carried out by solution methods and ispresented in Schemes 1 and 2. ##STR1## CF₃ CO₂ H.H-Gly-Val-Gly-OBzl(II): Boc-Gly-Val-Gly-OBzl (I) (10.0 g, 23.72 mmol) was stirred with 100ml of trifluoroacetic acid (TFA) for one hour and solvent removed underreduced pressure. The residue was triturated with ether, filtered,washed with ether and dried, to give 9.4 g (yield: 91%) of the deblockedpeptide.

Boc-Phe-Gly-Val-Gly-OBzl (III): Boc-Phe-OH (9.16 g, 34.54 mmol) wasdissolved in 100 ml of dimethylformamide (DMF), cooled to 0° C. andN-methylmorpholine (NMM) (3.83 ml) added. The solution was cooled to-15° C. and isobutyl chloroformate (IBCF) (3.96 ml, 30 mmol) was addedslowly under stirring while maintaining the temperature at -15°±1° C.After stirring at this temperature for 15 minutes, a precooled solutionof II (9.4 g, 21.59 mmol) and NMM (2.4 ml) in DMF (40 ml) was added andstirring continued for two hours at ice-bath temperature. A saturatedsolution of KHCO₃ was added to bring the pH to 8.0 and stirred for anadditional 30 minutes. The reaction mixture was poured into a cold 90%saturated NaCl solution (1000 ml) and the precipitate obtained wasfiltered, washed with satd. NaCl, H₂ O and dried. The peptide wascrystallized from ethyl acetate to give 10 g of III (yield: 81.5%).R_(f) ² 0.83 Anal. Calcd. for C₃₀ H₄₀ N₄ O₇ : C 63.35, H 7.09, N 9.85%.Found: C 62.96, H 7.37, N 9.86%.

Boc-Gly-Phe-Gly-Val-Gly-OBzl (IV): III (8.0 g, 14 mmol) was deblocked asdescribed for II and coupled with Boc-Gly-OH by the excess mixedanhydride method and worked up as described under III to obtain thetitle compound in 98.6% yield, R_(f) ² 0.75. Anal. Calcd. for C₃₂ H₄₃ N₅O₈ : C 61.42, H 6.92, N 11.19%. Found: C 61.12, H 7.21, N 11.19%.

Boc-Val-Pro-Gly-Phe-Gly-Val-Gly-OBzl (VI): Boc-Val-Pro-OH (V) (29) (1.89g, 6.02 mmol) and 1-hydroxybenzotriazole (HOBt) (0.92 g, 6.02 mmol) weretaken in DMF (20 ml), cooled with ice-salt freezing mixture and reactedwith 1(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)(1.15 g, 6.02 mmol) for 15 min. IV was deblocked as described under IIand an ice-cold solution of this deblocked peptide (3.5 g, 5.4 mmol),NMM (0.61 ml) in DMF (20 ml) was added to the above activated acidcomponent and stirred for two days at room temperature. Solvent wasremoved under reduced pressure. The residue was taken in chloroform andwashed with water, 20% citric acid, water, satd. NaHCO₃, water, driedover anhyd. MgSO₄ and solvent removed under reduced pressure to give thedesired peptide (3.4 g, yield 75.7%), R_(f) ¹ 0.27, R_(f) ² 0.71 Anal.Calcd. for C₄₂ H₅₉ N₇ O₁₀ : C 61.36, H 7.23, N 11.93%. Found: C 61.25, H7.66, N 12.08%.

Boc-Ala-Gly-OBzl (VII): This peptide was prepared by the mixed anhydridemethod as described for the synthesis of III in 81% yield, R_(f) ¹ 0.24,R_(f) ⁴ 0.92. Anal. Calcd. for C₁₇ H₂₄ N₂ O₅ : C 60.16, H 7.13, N 8.25%.Found: C 59.68, H 7.17, N 8.09%.

Boc-Ala-Gly-OH (VIII): VII (16.97 g, 50 mmol) was taken in glacialacetic acid (170 ml) and hydrogenated overnight in the presence of 10%Pd/C (1.7 g) as catalyst at 40 psi. The catalyst was filtered with theaid of celite and solvent removed under reduced pressure. The residuewas taken in satd. NaHCO₃ solution, extracted with EtOAC (3×), cooled,acidified to pH 2.0 and re-extracted with EtOAC (3×). The combined EtOACextracts were washed with satd. NaCl, and concentrated under reducedpressure. The residue was triturated with EtOAC: Pet. ether (1:1) andthe precipitate obtained was filtered, washed with pet. ether and dried(10.9 g, yield: 87.5%), R_(f) ⁴ 0 52. Anal Calcd. for C₁₀ H₁₈ N₂ O₅.1/2H₂ O: C 46.50, H 7.41, N 10.84%. Found: C 46.64, H 7.41, N 10.84%.

Boc-Ala-Gly-Val-Pro-Gly-Phe-Gly-Val-Gly-OBzl (IX): VIII was coupled withthe deblocked VI using EDCI/HOBt as described for the preparation of VIto give the desired peptide in 84.8% yield. R_(f) ² 0.76, R_(f) ³ 0.28.Anal. calcd. for C₄₇ H₆₇ N₉ O₁₂ : C 59.22, H 7.08, N 13.22%. Found C58.73, H 7.55, N 13.14%.

Boc-Ala-Gly-Val-Pro-Gly-Phe-Gly-Val-Gly-OH (X): The nonapeptidebenzylester (IX) was hydrogenated as described under VIII to obtain theacid in quantitative yield. R_(f) ³ 0 24. Anal. calcd. for C₄₀ H₆₁ N₉O₁₂ .H₂ O: C 55.09, H 7.16, N 14.3%. Found: C 54.69, H 7.16, N 13.81%.

HCO₂ H.H-Ala-Gly-Val-Pro-Gly-Phe-Gly-Val-Gly-OH (XI): X (20 mg, 0.023mmol) was stirred with 95-97% formic acid (0.6 ml) for 6 hours andconcentrated under reduced pressure. The residue was taken in dist.water and lyophilized to obtain the product in quantitative yield. R_(f)³ 0.65. This completes synthesis of the first permutation.

Boc-Ala-Gly-Val-Pro-OBzl (XIII): To obtain the second permutation,Boc-Val-Pro-OBzl (XII) was deblocked with TFA and coupled with VIII asdescibed under VI to obtain the protected tetrapeptide in 67.4% yield.R_(f) ² 0.80, R_(f) ⁴ 0.80 Anal calc. for C₂₇ H₄₀ N₄ O₇ : C 60.54, H7.52, N 10.46%. Found: C 60.80, H 7.53, N 10.56%.

Boc-Gly-Phe-Gly-Val-Gly-OH (XIV): IV was hydrogenated and worked up asdescribed for VIII to obtain the product in quantitative yield. R_(f) ³0.16. Anal. calcd. for C₂₅ H₃₇ N₅ O₈ .H₂ O: C 54.23, H 7.10, N 12.65%.Found: C 54.14, H 6.93, N 12.21%.

Boc-Gly-Phe-Gly-Val-Gly-Ala-Gly-Val-Pro-OBzl (XV): After deblocking XIIIwith TFA, the salt was coupled with XIV using EDCI/HOBt and worked up asfor the preparation VI. The title compound was obtained in 75.5% yield.R_(f) ² 0.57. Anal. calcd. for C₄₇ H₆₇ N₉ O₁₂ .2H₂ O: C 57.06, H 7.23, N12.74%. Found C 54.23, H 7.10, N 12.74%.

Boc-Gly-Phe-Gly-Val-Gly-Ala-Gly-Val-Pro-OH (XVI): The above peptide XVwas hydrogenated as described for the preparation VIII to obtain theproduct in quantitative yield. R_(f) ³ 0.22. Anal. calcd. for C₄₀ H₆₁ N₉O_(12:) C 55.66, H 7.12, N 14.60%. Found: C 55.86, H 7.23, N 14.14%.

HCO₂ H.H-Gly-Phe-Gly-Val-Gly-Ala-Gly-Val-Pro-OH (XVII): The peptide XVIwas treated with formic acid and worked up as described under XI. R_(f)³ 0.06. This completes the synthesis of the second permutation.

Results

Carbon-13 nuclear magnetic resonance spectra for the two nonapeptidesdemonstrated their purity. The elastin nonapeptide AGVPGFGVG (compoundXI) and its permutation GFGVGAGVP are both chemoattractants for elastinsynthesizing ligamentum nuchae fibroblasts. FIG. 1 shows the positivemigration of fibroblasts in response to a concentration gradient rangingfrom 10⁻¹² to 10⁻⁵ M nonapeptide. Maximal activity was at 10⁻⁹ M forAGVPGFGVG. Four such curves were run and all peaked at 10⁻⁹ M. Itspermutation GFGVGAGVP (compound XVII) was tested with a concentrationrange of 10⁻¹² to 10⁻⁵ (FIG. 2). Again the maximal response was at 10⁻⁹M. Again, four experiments were completed and in all 10⁻⁹ M representedmaximal activity. FIG. 3 shows the response of the fibroblasts to humanplatelet derived growth factor at 0.3, 3, 10, 20, 30, 40, 50, 60, 70,90, 300 and 3000 ng/ml. Maximum activity is at 30 ng/ml (1 nM). Thehexapeptide, VGVAPG, of tropoelastin was chemotactic for fibroblastswith the peak of activity at 10⁻⁸ M (FIG. 4). Two such tests were doneand both peaked at 10⁻⁸ M. The "checkerboard analysis" was performedwith the two nonapeptides to identify the fibroblast migration aschemotaxis. Peptide was added to the lower compartment, to the uppercompartment (with the cells) or to both compartments to set up apositive, a negative or zero gradient, respectively. Tables I and II forAGVPGFGVG and GFGVGAGVP show that the cells preferentially migrated inresponse to a positive gradient and not to a negative gradient or to nogradient. The experiments represented by the data in Tables I and IIwere repeated three times and two times, respectively. In all cases, thedata followed the patterns seen in Tables I and II. The data demonstratethat the peptides stimulated direct (chemotaxis) rather than random(chemokinesis) migration of fibroblasts and that the peptides are truechemoattractants. Relative to the positive control of 30 μg/ml PDGF, thenonapeptides stimulated the same level of migration as PDGF.

EXAMPLE 2 Materials and Methods: Source of Chemicals and Materials

Modified Medium 199, fetal bovine serum trypsinEDTA and antibioticantimycotic solution were purchased from GIBCO, Chagrin, OH. The sourcesof the polycarbonate and cellulose nitrate membranes were NucleoporeCorp., Pleasanton, Calif. and Millipore Corp., Bedford, Mass. Thehematoxylin stain was from Harleco of Gibbstown, N.J. and the Hemacolorstain was purchased from American Scientific Products, Stone Mountain,Ga. Human plasma fibronectin was purchased from Calbiochem, San Diego,Calif.

Cell culture and cell preparation

Bovine thoracic aorta endothelial cells were isolated by scraping with ascalpel blade. Harvested cells were washed by centrifugation in Medium199 with Hank's salt solution containing penicillin-streptomycin and 10%fetal bovine serum. After dissociation of cell aggregates by extrusionthrough a 27 gauge needle, cells were seeded in 25 cm² flasks atdensities of about 10⁴ cells/cm² and reached confluency in 3 to 4 days.Cells were grown in Medium 199, 10% fetal bovine serum and 5% Ryan'sgrowth supplement (Dr. U.S. Ryan, U. Miami, FL). When cells reachedconfluency, they were subcultured at a 1:3 split ratio, after removalfrom culture dishes with a Costar cell scraper. Cells were monitored forpurity to specific fluorescent staining of endothelial uptake byacetylated low density lipoprotein (Stein and Stein, 1980, Netland etal, 1985) labeled with1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine perchlorate(Dil-Ac-LDL, Biomedical Technologies, Stoughton, MA). All studies wereperformed on first to fifth passage cells. These early passage cells atone to two days past confluency were dissociated in 0.05% trypsin, 0.02%EDTA in Hanks Balanced Salt Solution for 3 minutes at 37° C.,centrifuged, washed 2 times and resuspended in Medium 199 without fetalcalf serum. The trypsin reaction was stopped with soybean trypsininhibitor at 1 mg/ml. Cell concentration was determined with ahemocytometer and adjusted to a final concentration of 10⁶ /ml

Chemotaxis Assay

Chemotactic response was assayed wtih a 30 hole multi-blind-well plateas an adaptation of the modified Boyden chamber with the double membranetechnique. A collagen coated 8μ polycarbonate, polyvinyl pyrrolidone(PVP) free, membrane superimposed on a 0.45μ cellulose nitrate membraneseparated the lower and upper compartments containing respectively 0.24ml of test peptide dissolved in Medium 199 and 0.37 ml of the cellsuspension. The collagen pretreatment was intended to enhance cellattachment to the polycarbonate membrane. In the checkerboard assay, todistinguish between directed cell migration (chemotaxis) and random celllocomotion (chemokinesis), the concentration gradient between the upperand lower chambers were abolished by adding the test peptide at variousconcentrations to the cell suspension. For all experiments medium alonein the bottom chamber served as the baseline control. Human plasmafibronectin at 100 μm/ml in the bottom was the positive control. Thefilled plate was incubated for 5 hours at 37° C. in a humidifiedincubator with 5% CO₂ -air. The membrane pairs were recovered, fixed inethanol, stained in Harris' alum hematoxylin, hydrated in a gradedseries of propanol and cleared in xylene. The membrane pairs weremounted on glass slides and cell locomotion quantitated in the volumebetween the two membranes using bright field at 400X. Five randomlychosen fields were counted per membrane pair with there being 3 membranepairs per experimental condition. The average of the 15 fields wascalculated as was the standard error of the mean (S.E.M.) where n was15. Net cell migration was plotted as a function of peptideconcentration with net migration being the average number of cellsmoving in response to the peptide minus the average number of cellsmoving in response to medium alone.

The chemotactic response was also assayed with the Neuroprobe (Bethesda,MD) micro blind-well chamber. The lower wells contained 30 μl of Medium199 ± test peptide of fibronectin, and the upper wells 55 μl of thecells suspended in Medium 199. A 8-μm pore size. PVP-free, rectangular,polycarbonate, gelatin and fibronectin treated, membrane separated theupper and lower chambers. The gelatin pretreated membrane was incubatedin a PBS solution of 100 μg/ml fibronectin at room temperature for 2hours, air dried and used within 2 weeks. Once the cells were added tothe assembled chamber, it was incubated at 37° C. in 95% air--5% CO₂ for4 hours. The filter was removed, the top layer of nonmigrating cellsscraped off and the bottom layer of migrating cells fixed in ethanol andstained with Hemacolor. Quantitation of cell movement was the same asfor the 30 well chamber. In the inventors' hands, both chemotaxischambers were equally effective as were both membrane treatments (i.e.,with and without a fibronectin coating on the gelatin).

Peptide Synthesis

The synthetic procedures were analogous to those discussed above.

Results

The carbon-13 nuclear magnetic resonance (CMR) spectra of the twononapeptides are give in FIG. 5, indicating the correctness of synthesisand the purity of the final products. FIG. 6 shows a phase contrastimage of a typical monolayer of bovine aortic endothelial cells, whichexhibit a "cobblestone" distribution pattern. After incubation wtihDil-Ac-LDL, cells showed a punctate fluorescence distribution pattern,indicative of endosomal localization of the labeled LDL derivativeinternalized by cells via receptor-mediated endocytosis. All cells werepositive in fluorescent labeling with respect to controls withvariability to cell to cell staining density typically present.

Elastin synthetic peptides are chemoattractants for bovine aorticendothelial cells. FIG. 7 presents the data collected in an effort todetermine the optimal concentration of maximal stimulation of theendothelial cell migration. The dose response curve for the elastinrepeat nonapeptide and early passage endothelial cells indicate thatmaximal activity is a 8×10¹⁰ M for both peptides, the Phe-containingnonamer (GFGVGAGVP) and the Leu-containing nonamer (GLGVGAGVP). Thesedose response experiments were repeated 10 times for the Phe-nonapeptideand 9 times for the Leu-nonapeptide; all had the same results includingone study which was counted blind (i.e., the slides were coded and thecode was unavailable to the microscopist). FIG. 7 represents the datafrom two different experiments. The data were normalized with respect tothe fibronectin positive control because of variability in cellresponsiveness from experiment to experiment. The left ordinate presentsthe results as a percent of the fibronectin positive control, the rightordinate gives the actual number of cells migrating per high power field(h.p.f.), for both the Phe and Leu containing nonapeptides. Note thedifferences in the scales, indicating that in the Phenonapeptideexperiment, the positive control was 30 cells per h.p.f. and in theLeu-nonapeptide experiment, the positive control was 46 cells per h.p.f.With the data normalized this way, it is evident that both nonapeptidespeak at the same concentration and elicit the same degree ofresponsiveness. The biphasic aspect of the curves may be due to severalprocesses. One is the possibility of saturation of the cell receptorsthat trigger the chemotactic response and the other is the possibilityof gradient breakdown.

Table III presents the data for the checkerboard assay. As expected forchemotaxis, the values along the diagonal indicate zero net cellmigration when the concentrations in the two chambers are equivalent,above the diagonal the migration is not significant whereas below thediagonal there is a concentration dependent net movement of cells. Thesedata establish that the repeat elastin nonapeptides promote chemotaxis(i.e., directed movement) rather than solely chemokinesis (i.e.,increased random movement). This experiment was repeated 7 times andeach experiment had the same pattern.

The hexapeptide of elastin also induced cell migration (FIG. 8).However, maximal activity shifted by over a decade of concentration to10⁻⁸ M. Relative to the fibronectin positive control, the hexapeptide isless active than the two nonapeptides. This experiment was repeated 7times and the same pattern was obtained.

                  TABLE III                                                       ______________________________________                                        Checkboard Analysis of GFGVGAGVP with                                         Endothelial Cells                                                             GFGVGAGV-                                                                     P (M), lower                                                                            GFGVGAGVP (M), upper compartment                                    compartment                                                                             0        10.sup.-10                                                                              8 × 10.sup.-10                                                                  10.sup.-8                                ______________________________________                                        0         0(16)    4 ± 2  1 ± 2                                                                              -3 ± 1                                10.sup.-10                                                                              6 ± 2 1 ± 2  3 ± 2                                                                              -1 ± 1                                8 × 10.sup.-10                                                                    19 ± 3                                                                              11 ± 1 -3 ± 1                                                                             3 ± 1                                 10.sup.-8 4 ± 2 -1 ± 2 3 ± 2                                                                              1 ± 1                                 ______________________________________                                    

Discussion

Peptide purity is an important issue for these chemotactic experiments;one reason is the sharpness of the concentration dependency. Ifimpurities were present from peptide synthesis and varied from lot tolot, inappropriate amounts would be weighed out for the cell migrationassay and the peak could easily be missed. Also the impuritiesthemselves could elicit a chemotactic response. The CMR spectra attestto the peptides' purity. As important as a pure chemo-attractant is tothe chemotaxis experiment, so too is a pure cell population thatresponds to a positive control as previous described. FIG. 6demonstrates the purity of the cell culture and FIG. 7 demonstrates thatthese endothelial cells do migrate towards fibronectin.

The results indicate that the elastin nonapeptides can serve aschemoattractants for endothelial cells. This finding has significance inthat the elastin synthetic peptide becomes one of few pure synthetic andchemically defined compounds both to support chemotaxis and to bereadily available and at reasonable cost. It also is easily handled,requiring no special storage and dissolution techniques. A wide varietyof factors can effect endothelial migration. Some are from blood:platelet factors, lymphocyte products, mitogenic factors from leukocytecultures, lymphokines, interferon, fibrinogen and its fragments. Manyfactors have been isolated from tissue in soluble form. These includetumor derived factors, angiogenic factors and preparations and growthfactors. There are data indicating that fibronectin alone and/or inconcert with gangliosides or heparin constitutes a chemoattractant forendothelial cells. From this rather extensive listing it is apparentthat, aside from fibronectin, fibrinogen and heparin, there are few pureand chemically defined substances that are both supportive of chemotaxisand readily obtainable. The results suggest the use of thesenonapeptides as new standards for endothelial cell chemotaxis.

The findings have potential relevance to development of biomaterials,especially in the area of vascular prostheses. Biomaterials thatcomprise current vascular prostheses do not adequately support thedevelopment of an endothelial lining. It is reasonable to suggest thatfuture prostheses will need to use biomaterials that support cellattachment, cell growth and migration of the components of the vascularwall with special emphasis on endothelial cells to improve the patencycharacteristics of synthetic small vessels. The nonapeptides couldprovide chemotactic stimuli for endothelial cell migration into thevascular prosthesis.

One of the first morphological events observed in angiogenesis is themobilization of capillary endothelium. Directed migration of chemotaxishas been proposed as one aspect of the process of angiogenesis andneovascularization. The elastin nonapeptide may be an angiogenic factorin that it promotes directed movement of endothelial cells. It maypromote the next step of causing capillaries to infiltrate tissues.

The invention now being fully described, it will be apparent to one ofordinary skill in the art, that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A prosthetic device wherein a surface of saiddevice has incorporated into said surface a chemotactic peptide of theformula

    B.sup.1 -X-(AGVPGLGVG).sub.n -(AGVPGFGVG).sub.m -Y-B.sup.2

wherein A is a peptide-forming residue of L-alanine; P is apeptide-forming residue of L-proline; G is a peptide-forming residue ofglycine; V is a peptide-forming residue of L-valine; F is apeptide-forming residue of L-phenylalanine; L is a peptide-formingresidue of L-leucine; B¹ is H or a biocompatible N-terminal group; B² isOH, OB³ where B³ is a non-toxic metal ion, or a biocompatible C-terminalgroup: X is GVPGFGVG, GVPGLGVG, VPGFGVG, VPGLGVG, PGFGVG, PGLGVG, GFGVG,GLGVG, FGVG, LGVG, GVG, VG, G or a covalent bond; Y is AGVPGFGV,AGVPGLGV, AGVPGFG, AGVPGLG, AGVPGF, AGVPGL, AGVPG, AGVP, AGV, AG, A or acovalent bond; n is an integer from 0 to 50; m is an integer from 0 to50; with the proviso that when both n and m are 0, X and Y are selectedso that the chemotactic peptide has at least 3 amino acid residues inthe X and Y positions combined.
 2. The device of claim 1, wherein n isfrom 1 to 10 and m is 0 to
 10. 3. The device of claim 1, wherein n isabout 5 and m is 0 to
 5. 4. The device of claim 1, wherein n is 1 and mis
 0. 5. The device of claim 1, wherein said peptide is H-GFGVGAGVP-OHor a salt thereof.
 6. The device of claim 1, wherein B¹ is H and B² isOH or OB³ where B³ is an alkali metal ion.
 7. The device of claim 1,wherein said amount is from 10⁻⁹ to 10⁻³ millimoles of nonamer ofrepeating unit per 100 cm² of said surface.
 8. The device of claim 1,wherein said prosthetic device comprises a structural polypeptide. 9.The device of claim 1, wherein said chemotactic peptide is incorporatedusing non-covalent bonding between said chemotactic peptide and saidsurface.